Interactions of human truncated DISC1 proteins: implications for schizophrenia

Abstract

Numerous genetic linkage and association reports have implicated the Disrupted-in-Schizophrenia (DISC1) gene in psychiatric illness. The Scottish family translocation, predicted to encode a C-terminus-truncated protein, suggests involvement of short isoforms in the pathophysiology of mental disorders. We recently reported complex alternative splicing patterns for the DISC1 gene and found that short isoforms are overexpressed in the brains of patients with schizophrenia and in carriers of risk-associated alleles. Investigation into the protein-protein interactions of alternative DISC1 isoforms may provide information about the functional consequences of overexpression of truncated forms in mental illness. Human embryonic kidney (HEK293) cells were transiently co-transfected with human epitope-tagged DISC1 variants and epitope-tagged NDEL1, FEZ1, GSK3β and PDE4B constructs. Co-immunoprecipitation assays demonstrated that all truncated DISC1 variants formed complexes with full-length DISC1. Short DISC1 splice variants LΔ78, LΔ3 and Esv1 showed reduced or no binding to NDEL1 and PDE4B proteins, but fully interacted with FEZ1 and GSK3β. The temporal expression pattern of GSK3β in the human postmortem tissue across the lifespan closely resembled that of the truncated DISC1 variants, suggesting the possibility of interactions between these proteins in the human brain. Our results suggest that complexes of full-length DISC1 with truncated DISC1 variants may result in cellular disturbances critical to DISC1 function.

Figure 1

(a) Epitope-tagged DISC1 constructs. Schematic representation of the epitope-tagged constructs coexpressed in HEK293 cells. The continuous line is indicative of the genomic sequence of DISC1 with the numbered exons shown as rectangles. An N-terminal Myc and/or FLAG epitope was added to all DISC1 constructs. Known DISC1 (L, Lv and S) and novel isoforms termed ‘DISC1 LΔ78T10' (deletion of exons 7 and 8; termination at exon 10), DISC1 LΔ78T9 (deletion of exons 7 and 8; termination at exon 9), DISC1 LΔ3 (deletion of exon 3) and Esv1 (insertion in exon 3) were used. (b) DISC1 self-association. Short isoforms bind to full-length DISC1. All FLAG-tagged DISC1 isoforms (Lv, LΔ3, LΔ78T10, LΔ78T9, S and Esv1) co-immunoprecipitate with myc-tagged, full-length DISC1 (L). Precipitated proteins were resolved on SDS-PAGE and immunoblotted with an antibody against FLAG DISC1. N=4 separate experiments. (c and e) Inputs used in co-immunoprecipitation. Lysates used in experiment were subjected to SDS-PAGE in parallel with the directly immunoprecipitated proteins. Anti-FLAG and myc immunoblots of inputs show comparable protein expression present during immunoprecipitation and bands at expected molecular weights. (d) Differential interactions of truncated DISC1 proteins. Essential proteins known to interact with full-length DISC1 (L) were examined for interaction with short isoforms. After coexpression of Myc-tagged DISC1 isoforms with FLAG-tagged DISC1-interactors, lysates were subjected to immunoprecipitation with anti-Myc DISC1 and immunoblotted with an anti-FLAG antibody. First panel: all DISC1 isoforms bind FEZ1. Second panel: DISC1 isoforms co-immunoprecipitate with GSK3B. Third panel: DISC1L and Lv interact with NDEL1. Fourth panel: DISC1LΔ3 and LΔ78 show reduced binding with PDE4B. N=3–4 separate experiments. (f) Quantification of DISC1 self-associations. Densitometry readings of immunoprecipitated bands were normalized to full-length DISC1L. Numbers indicate the coexpressed DISC1 isoforms (1: DISC1 Lv, 2: DISC1 LΔ3, 3: DISC1 LΔ78T10, 4: DISC1 LΔ78T9, 5: DISC1 S and DISC1 Esv1). (g) Quantification of DISC1 interactions with FEZ1, GSK3β, PDE4B and NDEL1. Densitometry readings of immunoprecipitated bands were normalized to input bands as follows 1; DISC1L, 2: DISC1 Lv, 3: DISC1 LΔ3, 4: DISC1 LΔ78T10, 5: DISC1 LΔ78T9, 6: DISC1 S and 7: DISC1 Esv1. Error bars show s.d. and asterisks are given at P<0.05.

Figure 2

mRNA expression of DISC1-binding partners across the lifespan. Expression of FEZ1 (a), GSK3β (b), NDEL1 (c) and PDE4B (d) mRNA was measured in the human dorsolateral prefrontal cortex across the lifespan using custom-spotted Illumina microarrays. Each dot indicates the expression level of an individual subject reported as log2 (sample/reference). The x axis is labeled in fetal weeks, (14–20) followed by postnatal life in years (0–80). The expression trajectory (blue line) represents loess fitting of the expression data.

Figure 3

Predicted binding sites of truncated DISC1 proteins. A diagram showing binding sites of full-length DISC1 (L) protein with NDEL1, PDE4B, GSK3β, FEZ1 and DISC1 self-association sites as determined by previous studies.^{, , , , ,} Black rectangles indicate areas of coiled coil-forming potential, and the dotted line signifies the translocation breakpoint of the Scottish family translocation. Shown below the full-length DISC1 form is a representation of the protein isoforms translated from novel variants DISC1 LΔ3, LΔ78T9, Esv1. In parenthesis, the source sequence of the transcript is listed according to the April 2011 version of the NCBI database. Along with exon deletions that may disrupt protein interactions, the short isoforms may lose C-terminus-binding sites.